Structural mechanism of signal transduction in a phytochrome histidine kinase

Phytochrome proteins detect red/far-red light to guide the growth, motion, development and reproduction in plants, fungi, and bacteria. Bacterial phytochromes commonly function as an entrance signal in two-component sensory systems. Despite the availability of three-dimensional structures of phytochromes and other two-component proteins, the conformational changes, which lead to activation of the protein, are not understood. We reveal cryo electron microscopy structures of the complete phytochrome from Deinoccocus radiodurans in its resting and photoactivated states at 3.6 Å and 3.5 Å resolution, respectively. Upon photoactivation, the photosensory core module hardly changes its tertiary domain arrangement, but the connector helices between the photosensory and the histidine kinase modules open up like a zipper, causing asymmetry and disorder in the effector domains. The structures provide a framework for atom-scale understanding of signaling in phytochromes, visualize allosteric communication over several nanometers, and suggest that disorder in the dimeric arrangement of the effector domains is important for phosphatase activity in a two-component system. The results have implications for the development of optogenetic applications.


DrBphP-DrRR.
(a) Size-exclusion chromatogram of DrBphP-DrRR prior to deposition on the grids for cryo EM, plotted at three phytochrome-specific wavelengths. The chromatogram indicates pure and monodisperse sample. (b) Dark reversion of DrBphP-DrRR fusion and DrBphP with added DrRR, 1 conducted as in the main text. External addition of excess DrRR (10 µM) to DrBphP (1 µM) increased the dark reversion rate of the phytochrome. However, this reversion rate was slower than with the fusion, which can be explained by high local concentration of DrRR in the DrBphP binding site of the fusion and by different buffer conditions used in Multamäki et al. 2021. 1 The decay times (with 95% confidence bounds and amplitude) were τ1 = 2.9 min (2.2 min, 4 min, 35%) and τ2 = 246 min (208 min, 301 min, 65%) for DrBphP; τ1 = 5.4 min (4.2 min, 7.7 min, 44%) and τ2 = 179 min (141 min, 244 min, 56%) for DrBphP-DrRR; and τ1 = 2.4 min (1.8 min, 3.4 min, 36%) and 216 min (184 min, 260 min, 64%) for DrBphP + DrRR. (c) Extended version of the gel shown in Figure 1d. The gel contains the same experiment with two different incubation times (10 min and 20 min). We observe that the amount of remaining p-DrRR depends on the incubation time, but that the relative phosphatase activities of each sample remain approximately the same. For source data, see separate Source Data file.

Supplementary Figure 2: Scheme for reconstruction of the electron density maps in Pr.
Summary of the reconstruction procedure from single-particle images. After ab initio reconstruction, class2 and class3 contain impurities and broken particles and were therefore discarded. The best 3D reconstruction class1 was used for further refinement steps. The final full-length 3D reconstruction of Pr state has an overall resolution of 3.6 Å. The local refinement with a mask of PSM and part of the neck region yields an improved overall resolution of 3.4 Å.

Supplementary Figure 3: Homology models indicate a potential wide conservation of the helical neck and comparison of cryo-EM to crystal structures.
(a-d) The "neck" is a conserved feature across the phytochrome superfamily. The reconstructed Pr state of DrBphP (Q9RZA4) (a) was compared to SWISS-MODEL homology models of cyanobacterial Cph1 (Q55168) (b), Fungal EnFphA (Q5K039) (c), and an AlphaFold model of plant AtPhyA (P14712) (d). All homologues possess a neck region with buried interface (shown with arrows), suggesting that the photoactivation mechanism may be conserved. (e-f) Comparison of the crystal structures of the photosensory module of DrBphP (Q9RZA4, pdb codes 4O0P for Pr and 4O01 for Pfr) overlaid with the structures obtained in this work indicate that the Pr crystal structure overlaps well (RMSD 1.330 for residues 22-502), but that the Pfr crystal structure diverges (RMSD 1.416 for residues 22-502). See arrows for largest discrepancies.

Supplementary Figure 4: Scheme for reconstruction of the electron density maps in Pfr.
Summary of the reconstruction procedure from single-particle images. After ab initio reconstruction, class3 was discarded. It contained incomplete particles and impurities. Class1 was similar to the full-length model of Pr from the experiment in the dark ( Supplementary Fig.  2), as it contained densities for a β sheet in the tongue region. Class2 gave a different full-length model compared to class 1 and showed a novel positioning of the output domains and helical densities in the tongue region. We assign this class to Pfr. For further refinement, we first pooled all particles from class1 and class2 for heterogeneous refinement, using class1 and class2 as 3D reference models. This essentially reassigns and realigns particles to the Pr and Pfr classes. A second run of heterogeneous refinement was performed on class 2 to further clean out Pr particles from the Pfr density. The final model of Pfr (3.9 Å resolution) had the hallmarks of a Pfr structure (see main text). By applying a mask over the PSM and part of neck, the Pfr PSM model was refined to 3.5 Å. Approximately 50% of the intact particles (class 1 and 2 after ab initio refinement) were in Pfr and 50% in Pr, close to the maximum turnover of the photoreaction of approximately 65%.

Supplementary Figure 5: Assignment of electron densities to CA and REC domains.
(a) Assignment of electron densities in Pr to CA and REC domains. The CA and REC domains are assigned as indicated on the map (blue with 50% transparency) from class 1 of the ab initio reconstruction step ( Supplementary Fig. 2). The modelled Pr structure (PAS-GAF-PHY-neck, blue) was fit into the density. (b) Assignment of electron densities in Pfr to CA and REC domains. The Pfr structures (PAS-GAF-PHY-neck, magenta) is shown with the Pfr electron density (magenta with 50% transparency) from class 2 of the ab-initio reconstruction step ( Supplementary Fig. 4). The density of the effector domains is asymmetric. We tentatively assign patches of density to the CA domains of both monomers and the REC domain of one of them as indicated. The neck helices are visualized from the top through the electron densities in both panels a and b. (c) Biliverdin in Pfr (magenta) and Pr (blue) superimposed with the electron density in Pr (blue). (d) Superimposed biliverdin in Pr (blue) and Pfr (magenta) with the electron density in Pfr (magenta).

Supplementary Figure 7: Local resolution and Fourier Shell Correlation of the maps in Pfr.
The sharpened cryo-EM densities of the Pfr state of (a) full-length and (b) PSM, colored based on local resolution produced using local resolution estimation in cryoSPARC. The Fourier shell correlation for the cryo EM maps of (c) full-length and (d) PSM are also shown. The resolution of map corresponds to FSC 0.143. Angular distribution calculated in cryoSPARC for particle projections of (e) full-length and (f) PSM. The heat maps show the number of particles for each viewing angle.
Representative micrographs collected from the grids illuminated with far-red (a) or red light (b). (c) The selected 2D class averages used for ab initio reconstruction step of the Pr state shown in Supplementary Fig. 2. (d) The selected 2D class averages after the second round of Topaz picking step of the Pfr state shown in Supplementary Fig. 4.